Perturbation of the Stability of Amyloid Fibrils through Alteration of Electrostatic Interactions

Abstract

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.

Single Point Mutations Induce a Switch in the Molecular Mechanism of the Aggregation of the Alzheimer’s Disease Associated Aβ42 Peptide

“…Conversely, the more extensive hydrogen bonded core and the higher levels of interfilament organization in I31E/E22G Aβ 42 are more likely to result in a more stable fibrillar state. 18,19,23 Correspondingly, we found that the I31E/E22G Aβ 42 fibrils are significantly more resistant to trypsin digestion than those of the E22G Aβ 42 peptide. Residues 17−28, which NMR studies have shown to be within the core of wild type Aβ 42 fibrils, 24,25 remained resistant to proteolysis by trypsin for greater than one hour for I31E/E22G Aβ 42 fibrils, whereas this same fragment is released from E22G Aβ 42 fibrils after only 20 min of tryptic digestion (Figure 2c).…”

Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy

“…Therefore, mutations within the core β-sheets have a higher potential of altering the alignment and arrangement of β-sheet packing and the extent of the hydrogen-bond network, which dictate the twist and rigidity of a single strand of protofilament. Because the packing of protofilaments within the fibril is stabilized by many weak long-range electrostatic (or hydrophobic) interactions along the protofilaments, all of which must accommodate the twist and rigidity of the protofilaments, changes in the β-sheet packing pattern and the stability of the β-sheet induced by a single mutation are likely to propagate to the supermolecular packing and the final twist of the fibrils (46)(47)(48). This result is demonstrated for mutations such as G37R and G41D, both of which occur within the N-terminal region that forms the fibril core and constitute significant changes in size as well as in charge.…”

Arresting Amyloid with Coulomb’s Law: Acetylation of ALS-Linked SOD1 by Aspirin Impedes Aggregation

“…The method that we used to determine the thermostability of acetylated and unacetylated apo-SOD1 fibrils was modified from Shammas and coworkers (32) and is illustrated in Fig. S15 A.…”